The project involves a comparative study of wild type and three temperature-sensitive (two cold-sensitive and one heat-sensitive) mutants of Neurospora crassa that have conditional defects in cytosolic ribosome biosynthesis in that each underproduces one of the ribosomal subunits at the nonpermissive temperature. The aim of the studies is to characterize the molecular bases for the defects of the mutant strains and thus obtain information about the mechanism and regulation of ribosome production in eukaryotes. Thus we shall study (a) whether the efects in the strains involve termperature-sensitive synthesis or function of the molecule responsible for the mutant phenotype; (b) the kinetics of synthesis and processing of pre-rRNA in the mutants; (c) the time point during pre-rRNA synthesis and processing at which the 3 mutant 's temperature-sensitive defects manifest themselves; (d) the RNA composition and processing of nuclear pre-ribosomal ribonucleoprotein particles in the mutants; (e) any quantitative differences in pre-rRNA synthesis and processing in the nuclei of the mutants; and (f) the methylases potentially involved in pre-rRNA methylation in wild type and the mutants. The project also involves studies on the regulation of macromolecular synthesis in the wild type. Work here will concentrate on the stable RNAs i.e. RRNA and tRNA, and the effect of media composition, media changes, temperature, temperature shifts on the synthesis of these RNAs and on the turn-over rate of rRNA. Finally during a sabbatical leave, work will be done to study the organization of the ribosomal DNA of Neurospora and the regions of the chromosome flanking it. (Techniques in these studies include density gradient centrifugation, acrylamide gel electrophoresis, nucleic acid extraction and purification, nuclei isolation and purification, radioisotope incorporation analysis.)